A. SPECIFIC AIMS The TGF- signaling system is involved in many developmental and physiological processes, including early embryogenesis, wound healing, inflammation, immunity and reproductive function. TGF- ligands form homoor heterodimers that interact with type I and type II receptor kinases, to form signaling complexes that influence cellular activation and cell fate decisions. In addition, extracellular regulators, such as follistatin, block TGF- signaling by interfering with ligand receptor interactions. While over 50 different TGF- ligands have been identified, these signal through a handful of type I (7) and type II (5) receptors and our quantitative understanding of how various receptorligand combinations are assembled and linked to differential physiological outputs remains incomplete. Several members of the TGF- family, including activins, GDF-9 and BMP-15, are critically important for normal reproductive function of the ovary, influencing key steps in oocyte development and release. GDF-9 and BMP-15 form a unique subset of the ligand superfamily, playing relatively specialized roles in ovarian development that contrast with the more widespread use of other TGF- ligands. Both GDF-9 and BMP-15 mutations have been linked to aberrant reproductive function, resulting in either significantly increased fertility or complete infertility. By virtue of their specialized usage within the reproductive axis and their central role in regulating ovarian follicle development, both GDF-9 and BMP-15 are attractive targets for potential therapeutic intervention and for use in the in vitro manipulation of follicle development. It remains to be established how GDF-9 and BMP-15 interact with their type I and type II receptors and how this may be regulated. The extracellular antagonist follistatin, which is a key modulator of activin signaling, may also play a role in the regulation of other TGF- ligands present during ovarian development. Follistatin can interact with a broad range of ligands, such as activins, BMPs and myostatin, and the mechanism by which it can engage such different ligands remains to be investigated. Follistatin exists in multiple isoforms that exhibit different interactions with heparan sulfate and TGF- ligands. We have previously focused on the interaction of activin A with its receptors and with follistatin, obtaining both structural and biochemical insights into this portion of the TGF- signaling system that is operative in the ovary. In order to gain a deeper understanding into the broader network of TGF- signaling pathways that are critical to normal ovarian development and physiology, and how these may be regulated by follistatin, we propose the following specific aims: Specific Aim 1: Regulation of follistatin 315 ligand interactions by its C-terminal region. Subaim 1.1 Test the functional role of follistatin D3 and the D2-D3 groove in follistatin 315. Subaim 1.2 Determine if the follistatin 315 C-terminal tail affects the solution conformation of follistatin, thereby reducing heparin and TGF- ligand binding affinity. Subaim 1.3 Determine the structures of the free FS288 and FS315 isoforms by X-ray crystallography. Specific Aim 2: Quantitative studies of follistatin interactions with activins, GDF-9 and BMP-15. Subaim 2.1 Measure the binding affinities of follistatin for TGF- ligands important in ovarian development. Subaim 2.2 Determine the role of the follistatin N-domain in binding to BMP-15, GDF-9 and other BMP molecules. Subaim 2.3 Establish whether TGF- ligand and heparin binding are coupled for BMP ligands similarly to activin A. Subaim 2.4 Provide a structural basis for understanding follistatin recognition of different TGF- ligands that use different sets of type I and type II receptors Specific Aim 3: Specificity and affinity of activins, GDF-9 and BMP-15 for type I and II signaling receptors. Subaim 3.1 Comparative analysis of activin, GDF-9 and BMP-15 interactions with ActRIIB and BMPRII receptors. Subaim 3.2 Investigation of activin, GDF-9 and BMP-15 interactions with Alk4, Alk5, Alk6 and Alk7 type I receptors. Subaim 3.3 Structural studies of GDF-9 and BMP-15 proteins alone and in receptor-bound complexes.